T-cell/Natural killer-cell neoplasms presenting as leukemia- Case series from single tertiary care center.

BACKGROUND
Mature T/ NK-cell neoplasms are a rare group of disorders and their presentation as leukemia is even rarer. Most of the previous studies have focused on mature B-cell lineage leukemias and there is a paucity of data on mature T/NK-cell lineage leukemias. We, therefore, planned this study to analyze their spectrum, frequency, morphology and immunophenotypic features.


SUBJECTS AND METHODS
All cases of lymphomas presenting as leukemia over a period of two and a half years were evaluated. Detailed analysis of cases with T/NK-cell lineage was done for their clinical, hematological and immunophenotypic features.


RESULTS
A total of 262 cases of mature lymphoid neoplasms presented as leukemia during the study period. Of whom, only 8 (3.1%) cases were of T /NK-cell lineage and the remaining (96.9%) were of B-cell lineage. Of 8 cases, 4 (50%) had T-prolymphocytic leukemia, 2 (25%) had chronic lymphoproliferative disorder- natural killer cell and 1 (12.5%) case of each T-large granular lymphocytic leukemia and hepatosplenic γ/δ T-NHL.


CONCLUSION
T/NK-cell leukemias are rare. Along with clinical and morphological features, pattern of immunophenotypic markers is vital for their diagnosis and subcategorization.


INTRODUCTION
Leukemias arising from mature Tlymphoid/natural killer (T/NK) cell lineage are relatively uncommon than those arising from Blineage. The incidence of T/NK-cell neoplasms in a large international study has been reported to be 10-15%; however, variation in incidence in different geographical regions and racial populations is documented. 1,2 The mature T-cell neoplasms result from clonal proliferation of post thymic T-cells, which can be either-α/β and γ/δ depending upon type of T-cell receptor (TCR) present on their surface. 3 α/β subtype is commoner, seen in 95% cases and γ/δ subtype accounts for <5% of mature T-cell. On the basis of the clinical manifestations and origins, mature T-lymphoid neoplasms are classified into three main groups: (i) primary leukemias which arise in the bone marrow (BM) and evolve with a leukemic phase; (ii) leukemia/lymphoma syndrome or the leukemic phase of T-cell lymphomas (T-NHL) which arises in the lymphoid tissues, but a leukemic picture is very 14 International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir common; and (iii) T-NHL, which originates in lymphoid tissues and rarely involves the blood. 4 Mature NK-cell neoplasms are characterized by persistent proliferation of NK-cells, which are derived from T-cells, but differ immunologically by the absence of TCR gene rearrangements and TCR protein. NK-cells thus express T-cell antigens but are negative for surface CD3; in addition, they also express NK-cell associated antigens-CD16, CD56 and CD57. Mature NK-cell neoplasms presenting as leukemia may have either an indolent course or may be aggressive. They comprise of: (i) chronic lymphoproliferative disorders of NK-cells (CLPD-NK), which have an indolent course and are characterized by persistent (>6 months) increase in peripheral blood (PB) NK-cells (usually ≥ 2x10 9 /L) without a clearly identified cause; 5 and (ii) aggressive NK-cell leukemias which have systemic neoplastic proliferation of NK-cells and are commonly associated with a leukemic blood picture. 6 The subtypes of mature T/NK-cell leukemias are outlined in Table 1. Most of the previous studies have focused on leukemias arising from mature B-cells and there is a paucity of data on those arising from mature T/NK. We, therefore, planned this study to analyze the spectrum, frequency, morphology and immunophenotypic features of mature T/NK-cell leukemias in our group of patients.

SUBJECTS AND METHODS
We analyzed 262 consecutive newly diagnosed cases of mature lymphoid neoplasms presenting as leukemia over a two and a half-year period (January 2011-June 2013). Detailed clinical data was obtained for all the patients. Findings of complete blood counts (performed on LH-750, Beckman Coulter, USA) and morphology of atypical lymphoid cells on PB and BM aspirate smears (May-Grunwald-Giemsa stained) were noted. BM biopsies were evaluated for percentage of cellularity and pattern of infiltration. Immunophenotyping was performed on PB and/or BM aspirate samples containing adequate number of atypical cells, using combination of fluoroscein isothiocynate (FITC), allophycocyanin (APC), phycoerythrin (PE) and peridinin chlorophyll protein complex (Per-CP) tagged monoclonal antibodies (MoAb). The primary panel included CD19, CD5, CD23, CD20, CD22, CD10, CD38, CD79b, CD43, CD200, FMC-7, kappa, and lambda, CD3, CD4 and CD8. Additional secondary panel was put when the primary panel did yield definite diagnosis and included CD2, CD7, CD1a, TdT, CD16, CD56, CD57, anti-TCR-α/β, anti-TCR-γ/δ, CD25, CD11c and CD103. Staining was done using lyse-wash technique; permeabilization was done for cytoplasmic and nuclear antigens after addition of MoAb for surface antigens. Immunophenotypic analysis was performed on FACS-CANTO-II (BD-Biosciences, San Jose) and a minimum of 10,000 events were analysed. A positive expression was defined based on antigen intensity, when 20% or more lymphoid cells showed fluorescence intensities in excess of the fluorescence intensity associated with the negative control. The cases were diagnosed and categorized based on the clinical details, morphology and immunophenotypic findings.

Clinical Features
There was a wide range in the age distribution, the youngest patient was a 22-year-old male with CLPD-NK cell and the oldest ones were a 63-year-old female and a male with T-PLL and T-LGL leukemia, respectively. The mean age was 42.5 years. There was a marked male predominance with a male to female ratio of 7:1. Organomegaly was observed in most of the cases. Hepatomegaly was present in all cases except in one case of T-LGL leukemia. Splenomegaly was also seen in majority of cases except in 1 case of T-PLL and T-LGL leukemia. All cases of T-PLL and hepatosplenic-γ/δ T-NHL presented with lymphadenopathy. It was absent in 1 case of CLPD-NK and T-LGL leukemia. Skin involvement and pleural effusion were seen in 1 case of T-PLL.

Hematological Features
Five of the 8 cases had hemoglobin < 10 gm/dL at the time of presentation, hemoglobin levels ranged from 4.5-13.3 gm/dL (median= 8.9 gm/dL). Thrombocytopenia was seen in all cases except in patient with T-LGL leukemia with a platelet count that ranged from 22-296 x 10 9 /L (median= 62 x 10 9 /L). Hyperleucocytosis was seen only in T-PLL cases (2 of 4 patients), overall total leucocyte count ranged from 7.5-235.1 x 10 9 /L (median= 13.9 x 10 9 /L). BM was hypercellular in all cases. Three of the 4 cases with T-PLL showed prolymphocytes with dense nuclear chromatin, conspicuous nucleolus and moderate amount of basophilic cytoplasm with blebbing in some of the cells. In one case of T-PLL, the prolymphocytes were smaller in size with cerebriform nucleus having inconspicuous nucleolus and basophilic cytoplasm with vacuolations (cerebriform variant of T-PLL) (Figure 1 a). Both cases of CLPD-NK showed intermediate-sized lymphocytes with condensed nuclear chromatin and moderate amount of cytoplasm with fine granules (Figure 1 b). The single case of T-LGL leukemia showed the presence of large atypical lymphocytes with moderate cytoplasm and coarse granules (Figure 1 c). A case of hepatosplenic γ/δ T-NHL showed atypical lymphoid cells with oval to round nuclei and moderate amount of cytoplasm (Figure 1 d). Bone marrow biopsy revealed diffuse and interstitial pattern of infiltration as most common pattern of infiltration. Intrasinusoidal infiltration was seen in 1 case of CLPD-NK cell and single case of hepatosplenic γ/δ T-NHL ( Figure 2). The details of clinical and hematological features are summarized in Table 2.

DISCUSSION
Amongst the non-Hodgkin's lymphomas, mature T/NK-cell neoplasms are less common than B-cell, and their presentation as leukemia is further infrequent. There is limited data on the mature T/NK-cell leukemias; however, in this study we analyzed their spectrum, frequency, morphology and immunophenotypic features. A total of 262 cases of mature lymphoid neoplasms presenting as leukemia were diagnosed at our center during the study period, of which only 8 (3.1%) cases were of International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir  T/NK-cell lineage. A previous study from India also reported a similar incidence of 4% in the cases analyzed by them. All cases in our study were adults. In our study, the median age was 42.5 years with a male predominance and our findings are consistent with previous studies. [7][8][9] WHO in its 2008 classification has outlined 18 subtypes for mature T/NK-cell neoplasms. 10 However; only few of them have a leukemic presentation, including T-PLL, T-LGL leukemia, Sezary Syndrome/Mycosis Fungoides (SS/MF), adult T-cell leukemia lymphoma (ATLL), CLPD-NK and aggressive NK-cell leukemia. In our study, half of the cases were of T-PLL which constituted 1.5% of total cases of mature lymphoid leukemias, which was similar to findings (2%) reported by Matutes et al. T-PLL is a disease of adults and affects mostly males, with median age of 65 years (range 30-94) at presentation. The principal disease characteristics are organomegaly, skin lesions and a raised lymphocyte count. In addition, skin infiltration and pleural effusion may also be present in some cases. Anemia and thrombocytopenia are common and lymphocyte count of >200 x 10 9 /L is seen in half of the cases. 11   The median age of our patients was 49 years (range= 31-63 years) with male predominance. Hepatomegaly and lymphadenopathy were seen in all cases, splenomegaly was present in 75% of cases and skin infiltration and pleural effusion were found in 1 case (25%) each. Thrombocytopenia and anemia were present in 100% and 75% of cases, respectively. Absolute lymphocyte count was > 200 x 10 9 /L in 50% of cases. In our study, 3 cases of T-PLL showed classical prolymphocytes morphology and 1 case showed prolymphocytes with cerebriform nucleus, inconspicuous nucleolus and basophilic cytoplasm with vacuolations. Three morphological variants have been reported in T-PLL. In 75% cases of T-PLL, the lymphocytes have condensed chromatin, regular or irregular nuclear outline, prominent nucleolus and basophilic cytoplasm with blebs (typical T-PLL). In the remaining, the prolymphocytes are smaller in size and the nucleolus is inconspicuous (small-cell variant T-PLL) or has a cerebriform nucleus (cerebriform variant of T-PLL). Immunophenotypically, 50% of our T-PLL cases were CD4+/CD8-and 50% were CD4+/CD8+. Most commonly-T-helper (CD4+ CD8-) phenotype is seen in T-PLL, in two-third of T-PLL cases. Remaining 25% may co-express CD4 and CD8 and cases with CD8+ CD4-are rare. A post-thymic T-cell phenotype with TdT negative, CD2+, CD5+, CD7+ and CD3+ is characteristic of T-PLL. 12 Although in this study, we did not find any case of ATLL and SS, they need to be considered in the differential diagnosis of T-PLL and T-PLL needs to be morphologically distinguished from B-PLL. ATLL and SS have overlapping morphological features and mature T-cell phenotype. Distinction amongst these is based on a constellation of clinical and laboratory features which include cell morphology, histology and immunophenotype. As cytoplasmic basophilia (more in T-prolymphocytes) and nuclear irregularity (irregular in half of the cases of T-PLL). The total leukocyte count is generally below 100 x 10 9 /L and massive splenomegaly is uncommon in ATLL and SS. Other features of differentiation include erythroderma in SS and hypercalcemia and positivity for HTLV-I in ATLL. Morphologically, SS is characterized by cells with cerebriform nuclei and ATLL in the presence of flower cells which have polylobated nuclei. They have some distinctive immunophenotypic features, i.e., SS is negative for CD7 and CD25, ATLL is positive for CD25 and negative for CD7 and T-PLL is positive for CD7 and negative for CD25. In our study, CLPD-NK cell (the second subtype) was seen in 2 patients. CLPD-NK is characterized by persistent increase in peripheral blood NK cells and was included as a provisional entity in the 2008 WHO classification. 5 Persistence of NK lymphocytosis for >6 months is important as a transient increase in circulating NK cells may be seen in conditions like autoimmune disorders or viral infections. CLPD-NK occurs predominantly in adults with no sex predilection. The majority of patients are asymptomatic, but some may have systemic symptoms or cytopenias, organomegaly and skin lesions are infrequent. 13 Both cases of CLPD-NK were seen in males, one was 22 years of age and the other one was 41 years. At the time of presentation, both had anemia, thrombocytopenia and hepatosplenomegaly. Morphologically, both cases had intermediate-sized lymphocytes with condensed nuclear chromatin and moderate amount of granular basophilic cytoplasm. Circulating cells in CLPD-NK are typically intermediate in size with round nuclei with condensed chromatin and moderate amounts of slightly basophilic cytoplasm containing fine or coarse azurophilic granules. 5 Such large granular lymphocytes may also be seen in T-LGL. The WHO classification recognizes 3 distinct disorders of large granular lymphocytes: T-LGL leukemia, CLPD-NK and aggressive NK-cell leukemia. 14 T-LGL is characterised by expansion of CD3+ CD8+ cytotoxic T-cells and CLPD-NK by expansion of CD3-cells. Other markers which help to identify NK cell lineage include CD16, CD56, CD57, KIR (CD158), CD94/NKG2A, CD161 and cytotoxic markers for TIA-1, granzyme B, granzyme M. Bone marrow infiltration may be interstitial or intrasinusoidal and immunohistochemistry may be needed to highlight the infiltration. In one of our cases, the infiltration was interstitial and in the other one both interstitial and intrasinusoidal pattern was seen. In this study, a 63-year-old male patient was diagnosed with T-LGL leukemia. He had organomegaly, anemia, easy fatigability and fever. Peripheral blood showed 81% LGL which were positive for CD3 and CD8. The trephine biopsy showed a nodular pattern of infiltration. T-LGL like CLPD-NK has an indolent clinical course, one-third of patients are asymptomatic and detected following an incidental finding of cytopenias on a routine blood test. It arises more commonly in patients with autoimmune disorders, particularly rheumatoid arthritis. Most LGL leukemias (80-90%) are CD3 positive with co-expression of CD8, CD16 and CD57, with CD56 and CD28 being negative. TCRα/β is usually seen and downregulation of CD5 and CD7 may occur. [15][16][17] A single case of hepatosplenic-γ/δ was seen in a 26year-old male who presented with fatigue, hepatosplenomegaly, lymphadenopathy, anemia and thrombocytopenia. Hepatosplenic T-NHL is an aggressive peripheral T cell lymphoma expressing γ/δ T cell receptors. It has male predominance and occurs predominantly in young adults with median age of 35 years. 18 Clinically, patients usually have marked splenomegaly and hepatomegaly may be seen in some cases, while lymphadenopathy is uncommon. Patients usually manifest marked thrombocytopenia, often with anemia. BM is involved with an intrasinusoidal pattern; 19 however, PB involvement is uncommon at presentation, but may occur later. Neoplastic cells are CD3+, TCRγ/δ+, CD56+/-, CD4-, CD8-/+ and CD5-. 18 Hepatosplenic-γ/δ T-NHL needs to be distinguished from (i) other NHL subtypes which show intrasinusoidal pattern of BM infiltration including SMZL, intravascular lymphoma, immunophenotyping is useful in differentiation and (ii) other lymphomas with γ/δ T-cells including primary cutaneous γ/δ T-cell lymphoma and extranodal T-cell NHL; however, unlike International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir hepatosplenic γ/δ, they involve extranodal sitesskin and subcutaneous tissue and intestine or nasal regions, thereby making this distinction easy.

CONCLUSION
Mature T/NK neoplasms presenting as leukemias constituted 3.1% of all mature lymphoid leukemias in this study. They are seen in adults with male predominance. Most patients had organomegaly, anemia and thrombocytopenia at presentation. Morphology of atypical lymphoid cells showed characteristic features. However, immunophenotyping with limited number of MoAb did not identify the subtype accurately, as there was overlap between the immunophenotypic profiles of different subgroups. Therefore, extended panel of markers including both T-lineage and NKlineage markers is required in conjunction with clinical details, cell morphology and histology for the diagnosis.

CONFLICT OF INTEREST
There is no conflict of interest to declare.